Animal models of corneal angiogenesis and corneal ectatic diseases, methods of producing, and methods of use thereof

ABSTRACT

Methods of producing non-human animal models of corneal angiogenesis and corneal ectatic diseases, such as corneal keratoconus, by applying an aromatic compound to the eye of a non-human animal are described. Also described are non-human animal models of corneal angiogenesis and corneal ectatic diseases, and methods of using the non-human animal models to screen compounds that modulate corneal angiogenesis and corneal ectatic diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Section 371 of International Application No.PCT/IB2015/000822, filed Jun. 3, 2015, which was published in theEnglish language on Dec. 10, 2015 under International Publication No. WO2015/185977 A1, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 62/007,140, filed Jun. 3, 2014, andthe disclosures of which are herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to non-human animal models of corneal diseases,and in particular animal models of corneal angiogenesis and cornealectatic diseases, such as corneal keratoconus.

BACKGROUND OF THE INVENTION

Angiogenesis is the physiological process by which new blood vesselsform from preexisting blood vessels. The concept of inhibitingangiogenesis as a therapeutic strategy, particularly for treating tumorsand cancers, has been discussed for several decades, and is now widelyconsidered to be a promising approach for the treatment of a range ofpathologies and disease states in which vascular proliferation is acomponent. Anti-angiogenesis strategies have now been pursued not onlyas anti-cancer therapies, but also for the treatment of arthritis,retinopathies, heart disease, and circulatory problems. Accordingly,experimental animal models of angiogenesis are important for studyingangiogenesis and the growth of blood vessels, evaluating the effects ofdifferent compounds on angiogenesis, and for screening compounds toidentify compounds having anti-angiogenic or pro-angiogenic activity.

The cornea has been considered an ideal model of in vivo angiogenesisbecause it is avascular, and therefore any vascular development (i.e.,development of new blood vessels) can usually be directly attributed toa substance or compound applied to the corneal area of the eye.Therefore, many animal models developed to study in vivo angiogenesisare models of corneal angiogenesis. These animal models are commonlyproduced by introducing a cornea pocket, or iris implant, into the eyeof an animal.

In the cornea pocket model, an inducer of angiogenesis, such as tumortissue, a cell suspension, or growth factor is placed into a pocketformed in the cornea, which induces the formation of new blood vessels.However, formation of the cornea pocket is often a difficult procedure,typically performed by lamellar dissection with a scalpel to create aspace or “pocket” in the cornea, into which the inducer of angiogenesisis introduced. Due to the surgical nature of the procedure,complications often result from the procedure including problems relatedto the anesthetic agent used, perforation of the anterior chamber of theeye during dissection, inadequate preparation of the inducer ofangiogenesis, and angiogenesis resulting from the surgical wound itselfor sutures used to stitch the surgical wound. Moreover, inflammatoryreactions can occur due to tissue manipulation and suturing, as well asin response to the inducer of angiogenesis inserted into the corneapocket, which is often a foreign substance. Reactions to the insertionof foreign materials in the eye can also cause fibrosis, which is theformation of excess fibrous connective tissue in an organ or tissue.Fibrosis is usually the result of a reparative or reactive process.

Moreover, to the best of the inventor's knowledge, there are currentlyno animal models of corneal ectatic diseases, such as cornealkeratoconus. Corneal ectasia is the progressive bulging of the corneadue to thinning or weakening of the cornea, accompanied by visiondeterioration, vision impairment, or both. Corneal keratoconus is one ofthe more common corneal ectatic diseases, and is characterized by astructural distortion of the cornea from the typical rounded shape to aconical shape that protrudes, or bulges, outward from the corneal areaof the eye. Animal models of corneal ectatic diseases would providetools for studying these diseases in vivo.

BRIEF SUMMARY OF THE INVENTION

Accordingly, there exists a need for new animal models of in vivoangiogenesis that overcome some of the disadvantages associated withprior art models of angiogenesis. There is also a need for animal modelsof corneal ectatic diseases that could be used to study these diseases.The invention satisfies this need by providing animal models of cornealangiogenesis that overcome certain disadvantages associated with priorart animal models of in vivo angiogenesis. The invention also providesanimal models of corneal ectatic diseases, such as corneal keratoconus.

In one general aspect, the invention relates to a method of producing anon-human animal model of corneal angiogenesis, the method comprisingapplying to a cornea of at least one eye of a non-human animal aneffective amount of an aromatic compound of formula (I):

wherein R is selected from the group consisting of hydroxyl, halogen,alkyl, alkoxy, and amino and n is 0, 1, 2, 3, 4, 5, or 6.

In a particular embodiment of a method of producing a non-human animalmodel of corneal angiogenesis according to the invention, an effectiveamount of the aromatic compound of formula (I) administered is about 1μmol to about 70 μmol.

In another general aspect, the invention relates to a method ofproducing a non-human animal model of a corneal ectatic disease, themethod comprising applying to a cornea of at least one eye of anon-human animal an effective amount of an aromatic compound of formula(I):

wherein R is selected from the group consisting of hydroxyl, halogen,alkyl, alkoxy, and amino, and n is 0, 1, 2, 3, 4, 5, or 6.

In a particular embodiment of a method of producing a non-human animalmodel of a corneal ectatic disease according to the invention, aneffective amount of the aromatic compound of formula (I) is about 30μmol to about 85 μmol.

In yet another general aspect, the invention relates to a method ofscreening compounds to modulate corneal angiogenesis or a cornealectatic disease, the method comprising:

-   -   (i) preparing a non-human animal model of corneal angiogenesis        or a corneal ectatic disease by a method comprising applying to        a cornea of at least one eye of a non-human animal an effective        amount of an aromatic compound of formula (I):

-   -   (ii) administering a test compound to the at least one eye of        the non-human animal model; and    -   (iii) determining an effect of the test compound on at least one        of blood vessel growth in a cornea and structural distortion of        a cornea in the at least one eye,        wherein R is selected from the group consisting of hydroxyl,        halogen, alkyl, alkoxy, and amino, and n is 0, 1, 2, 3, 4, 5, or        6.

Other aspects of the invention relate to non-human animal models ofcorneal angiogenesis and corneal keratoconus produced by the methods ofthe invention.

In particularly preferred embodiments of the invention, an aromaticcompound of formula (I) is benzene or phenol.

In yet another general aspect, the invention relates to a method ofidentifying substances harmful to human health, the method comprising:

-   -   (i) applying a test substance to a cornea of at least one eye of        a non-human animal; and    -   (ii) determining an effect of the test substance on at least one        of blood vessel growth in the cornea and structural distortion        of the cornea.

The details of one or more embodiments of the invention are set forth inthe description below. Other features and advantages will be apparentfrom the following detailed description, the drawings, and the appendedclaims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings embodiments which are presentlypreferred. It should be understood, however, that the invention is notlimited to the precise arrangements and instrumentalities shown in thedrawings and described in the following detailed description of theinvention. In the drawings:

FIGS. 1A, 1B, and 1C show photographic images of the right eye of aWistar rat before and after treatment with a 3.0 M phenol solution; FIG.1A: photographic image of the right eye prior to application of phenol;and FIG. 1B: photographic image of the right eye 3 weeks after a singletopical application of 3.0 M phenol solution (10 μl), and an insetshowing a schematic representation of the observed growth of bloodvessels in the cornea; FIG. 1C: image of a section of a cornealepithelium membrane stained with hematoxylin and eosin stain eight weeksafter a single topical application of 3.0 M phenol solution (10 μl);

FIGS. 2A, 2B, and 2C show photographic images of the right eye of Wistarrats treated with a single application of benzene solution of varyingconcentrations 15 days after application of the benzene solution; FIG.2A: eye treated with 4.0 M benzene solution (10 μl); FIG. 2B: eyetreated with 6.0 M benzene solution (10 μl); and FIG. 2C: eye treatedwith 7.0 M benzene solution (10 μl); and

FIG. 3 shows a photographic image of an eye of a Wistar rat 15 daysafter application of a 7.0 M aqueous benzene solution.

DETAILED DESCRIPTION OF THE INVENTION

All patents and publications referred to herein are incorporated byreference. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood to one ofordinary skill in the art to which this invention pertains. Otherwise,certain terms used herein have the meanings as set forth in thespecification.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural references unless thecontext clearly dictates otherwise.

As used herein, the term “non-human animal” refers to any animal, mostpreferably a mammal, which is not a human. Examples of mammals includecows, dogs, cats, horses, pigs, monkey, sheep, and rodents. Examples ofrodents include rats, mice, rabbits, and guinea pigs. Preferably, anon-human animal is a rodent selected from the group consisting of rats,mice, rabbits, and guinea pigs, and is more preferably a rat.

The term “cornea,” as used herein, refers to the transparent tissue atthe front of the eye that covers the iris, pupil, and anterior chamber.The cornea is avascular, meaning that it normally has no blood vessels.The cornea also typically has a rounded shape.

As used herein, “angiogenesis” and “neovascularization” refer to thephysiological process by which new blood vessels form from preexistingblood vessels. As used herein, the terms “corneal angiogenesis” and“corneal neovascularization” refer to the growth of one or more newblood vessels in the cornea. Because the cornea is avascular, i.e., doesnot contain any blood vessels, any new blood vessels in the corneatypically arise from the growth of blood vessels from the limbalvascular plexus area of the eye into the cornea.

As used herein, “fibrosis” refers to the formation of fibrous connectivetissue in an organ or tissue, usually as a result of a reparative orregenerative process. The fibrous connective tissue can be scar tissue.

As used herein, “corneal ectasia” and “corneal ectatic disease” refer toa noninflammatory disease of the cornea characterized by irregularitiesin the cornea that cause disturbances in vision as a result ofastigmatism. Corneal ectasia refers to a group of conditions includingkeratoconus, pellucid marginal degeneration, keratoglobus, and posteriorkeratoconus, with the most prevalent, particularly in humans, beingkeratoconus.

According to a preferred embodiment of the invention, a corneal ectaticdisease is keratoconus. The term “corneal keratoconus” refers to adisease that affects the structure of the cornea. In cornealkeratoconus, the shape of the cornea slowly changes from the typicalround shape to a conical shape that bulges outward, forming aprotrusion. Corneal keratoconus can also be described as “the loss ofshape” of the cornea.

As used herein, “structural distortion,” when used with reference to thecornea, means a change in shape of the cornea characterized by a bulgingor protruding of the cornea. In a particular embodiment, “structuraldistortion” refers to a change in shape of the cornea from a roundedshape to a conical shape that protrudes outward from the eye.

As used herein “an effective amount” refers to an amount of a compoundof formula (I) needed to induce the desired physiological result. In oneembodiment, an effective amount is an amount that induces the growth ofone or more new blood vessels in the cornea. In another embodiment, aneffective amount is an amount that causes a structural distortion of thecornea.

As used herein, the term “alkyl” means a saturated, unbranched orbranched hydrocarbon chain containing at least one carbon atom,preferably 1-20 carbons, and more preferably 1-3 carbon atoms. Examplesof unbranched alkyl groups include, but are not limited to, methyl,ethyl, propyl, butyl, pentyl, and hexyl. Examples of branched alkylgroups include, but are not limited to, isopropyl and tert-butyl.

As used herein, the term “alkoxy” denotes a unit having the generalformula —OR, wherein R represents an alkyl group. Examples of alkoxygroups include, but are not limited to, methoxy, ethoxy, propoxy, andbutoxy.

As used herein, the term “halogen” has its ordinary meaning as would beknown to one of ordinary skill in the art. Non-limiting examples ofhalogens include fluoro, bromo, chloro, and iodo.

An alkyl group can be unsubstituted or substituted with one or moresuitable substituents. When an alkyl group is substituted, it can haveone or more substituents, preferably from 1 to 3 substituents, and morepreferably from 1 to 2 substituents. Representative examples of suitablesubstituents with which an alkyl group can be substituted include, butare not limited to, halogens, such as fluoro, chloro, bromo, and iodo;hydroxyl; alkoxy, such as methoxy, ethoxy, and propoxy; and amino.

The invention relates to non-human animal models of corneal angiogenesisand corneal ectatic diseases, such as corneal keratoconus. The non-humananimal models are produced by applying an aromatic compound, such asbenzene or phenol, to an eye of a non-human animal. The inventorsurprisingly discovered that depending on the amount of aromaticcompound applied to the eye, corneal angiogenesis and/or corneal ectaticdiseases can be induced. For example, lower amounts of aromaticcompounds induce corneal angiogenesis, whereas increased amounts ofaromatic compounds are typically needed to induce corneal ectaticdiseases.

In one general aspect, the invention relates to a method of producing anon-human animal model of corneal angiogenesis. According to embodimentsof the invention, the method comprises applying to at least one eye of anon-human animal an effective amount of an aromatic compound of formula(I):

wherein R is selected from the group consisting of hydroxyl, halogen,alkyl, alkoxy, and amino and n is 0, 1, 2, 3, 4, 5, or 6.

According to preferred embodiments of the invention, the aromaticcompound of formula (I) is benzene or phenol. Benzene is an aromaticcompound of formula (I), wherein n is 0. Phenol is an aromatic compoundof formula (I), wherein R is hydroxyl and n is 1. In a particularlypreferred embodiment, the aromatic compound is phenol.

An effective amount of the aromatic compound of formula (I) can beapplied to the eye of a non-human animal by any method known in the artincluding, but not limited to, topical application and injection.Preferably, an effective amount of the aromatic compound of formula (I)is administered topically. Examples of topical compositions that can beused with the invention include, but are not limited to, creams, gels,ointments, and liquid compositions, such as solutions, suspensions, andeye drops. Preferred topical compositions include liquid compositions,and particularly eye drops.

According to embodiments of the invention, a compound of formula (I) canbe topically applied to the cornea by any method known in the art inview of the present disclosure. Non-limiting examples of methods fortopically applying a compound of formula (I) include by drops and byswabbing. Preferably, the compound of formula (I) is administered to thecornea by drops.

In a preferred embodiment of the invention, an aromatic compound offormula (I) is topically applied to the eye in a liquid composition, andmore preferably in the form of an aqueous vehicle. As used herein, an“aqueous vehicle” is a liquid composition comprising water and anaromatic compound of formula (I). Non-limiting examples of aqueousvehicles include solutions, suspensions, eye drops and the like. In aparticular embodiment of the invention, an aqueous vehicle containswater and benzene or phenol.

According to embodiments of the invention, the aromatic compound offormula (I) is applied to the cornea of the eye, and is preferablyapplied directly to the center of the cornea. The aromatic compound offormula (I) can be applied to one eye, or to both eyes of the animal. Incertain embodiments of the invention, applying the aromatic compound offormula (I) to only one eye of the animal is preferred, because thisallows for the second, untreated eye to serve as a control.

According to embodiments of the invention, an effective amount of acompound of formula (I) needed to produce a non-human animal model ofcorneal angiogenesis is about 1 μmol to about 70 μmol, and morepreferably 30 μmol to 70 μmol, such as about 1 μmol, 5 μmol, 10 μmol, 30μmol, 40 μmol, 50 μmol, 60 μmol, or 70 μmol. An effective amount of acompound of formula (I) can be applied once, or more than once.Preferably, an effective aromatic compound of formula (I) isadministered in a single application.

Because there is a difference in the angiogenic response betweendifferent animal species, and sometimes between animals of the samespecies, the effective amount of an aromatic compound of formula (I) toapply to an eye of the animal to induce corneal angiogenesis can dependon various factors including, but not limited to, the particular speciesof animal, the age of the animal, etc. One of ordinary skill in the artwould readily be able to determine an effective amount of an aromaticcompound of formula (I) to be applied to the eye of the non-human animalin order to achieve the desired physiological response in view of thepresent disclosure. One of ordinary skill in the art will alsoappreciate that there can be variability between animals of the samespecies in the angiogenic response to an aromatic compound of formula(I).

Any method known in the art can be used to evaluate the angiogenicactivity of any particular amount of an aromatic compound of formula (I)to determine if such amount is effective for inducing the desired amountof corneal angiogenesis. For example, the potency of angiogenic activitycan be evaluated by determining the number or growth rate of newlyformed capillaries and blood vessels, or by calculating an angiogenicscore. An angiogenic score can be calculated according to the followingformula: (vessel density)×(distance from the limbus). The limbus is theborder of the cornea and the sclera (white of the eye). A vessel densityvalue of 1 corresponds to about 0-25 vessels per cornea; a value of 2corresponds to about 25-50 vessels per cornea; a value of 3 correspondsto about 50 to 75 vessels per cornea; a value of 4 corresponds to about75-100 vessels per cornea; and a value of 5 corresponds to more than 100vessels per cornea. The distance from the limbus can be measured (in mm)with the aid of an ocular grid.

As another illustrative example, angiogenic activity can be evaluated byhistological studies. For examples, hematoxylin and eosin (H&E) stainscan be used to stain dissected corneal tissue, and detect the formationof new blood vessels. As shown in FIG. 1C, which is an image of asection of a corneal epithelium membrane stained with H&E stain eightweeks after a single topical application of a 3.0 M phenol solution,different biological structures can be detected to assess angiogenicactivity and the progression of corneal angiogenesis. In particular,arrow 1 points to normal corneal epithelium, arrow 2 points to new bloodvessels growth induced by the application of phenol that would notnormally be present in the cornea, and arrow 3 points to the normalstromal tissue of the cornea.

As an illustrative and non-limiting example, when the aromatic compoundof formula (I) is benzene or phenol, an effective amount can range fromabout 1 μmol to about 70 μmol (i.e., about 75 μg to about 5.5 mg), andis preferably from 30 μmol to 70 μmol, such as 1 μmol, 5 μmol, 10 μmol,30 μmol, 40 μmol, 50 μmol, 60 μmol or 70 μmol.

In a particular embodiment, the non-human animal used in a method of theinvention is an albino animal. As used herein, an “albino animal” refersto an animal lacking pigment, and particularly melanin, in at least theeyes. Albino animals produce purer animal models of corneal angiogenesisas compared to pigmented animals, which have melanin in their eyes,because pigmented animals tend to have a greater degree of cornealfibrosis upon application of aromatic compounds of formula (I), andparticularly benzene, to the cornea to induce angiogenesis.

The invention also relates to a non-human animal model of cornealangiogenesis produced by a method of the invention. According toembodiments of the invention, non-human animal models of cornealangiogenesis have substantially no fibrosis in the cornea of the eye.Additionally, the non-human animal models of corneal angiogenesiscomprise one or more new blood vessels in the cornea of at least one eyeof the animal. Because fibrosis is a natural biological process that canoccur in tissue repair, some fibrosis can be observed in the cornea ofnon-human animal models of the invention. However, the amount offibrosis observed, if any, is significantly less than the amount offibrosis that is observed with other animal models of cornealangiogenesis.

Non-human animal models of corneal angiogenesis thus have severaladvantages as compared to prior art animal models of cornealangiogenesis. In particular, due to the substantial absence of fibrosisin the cornea, a purer animal model of corneal angiogenesis is produced.More specifically, prior art animal models of corneal angiogenesis havea complicated angiogenic response, and in addition to the growth of newblood vessels, fibrosis or scar tissue formation is usually alsoobserved in the cornea. The presence of fibrosis and scar tissue makesit difficult and more complicated to understand the angiogenesisprocess. In contrast, the substantial elimination, or at leastsignificant reduction in the amount of fibrosis observed with thenon-human animal models of corneal angiogenesis of the invention allowsthe angiogenesis process to be more easily studied.

Moreover, less fibrosis and scar tissue formation is observed whenphenol is used as compared to the amount observed when benzene is used.Thus, phenol is the preferred aromatic compound of formula (I) for usewith the present invention.

In addition to a substantially reduced amount of fibrosis in the cornea,the non-human animal models of corneal angiogenesis according to theinvention have other advantages over prior art animal models. Forexample, the animal models can be produced by simply administering eyedrops of a solution of an aromatic compound of formula (I), such asbenzene or phenol, to the eye of the non-human animal. Accordingly,surgical procedures are not required. This eliminates many of thecomplications that are often associated with or result from surgicalprocedures, such as inflammatory reactions due to tissue manipulation,infection at incision sites, and puncturing of the anterior chamber ofthe eye. Moreover, suturing is not required, which further reducescomplications. Thus, the methods of producing non-human animal models ofthe invention are simpler to execute, less expensive, and require lesstime than previous methods for generating animal models of cornealangiogenesis, such as the corneal pocket model.

In another general aspect, the invention relates to a method ofproducing a non-human animal model of a corneal ectatic disease.According to embodiments of the invention, the method comprises applyingto a cornea of at least one eye of a non-human animal an effectiveamount of an aromatic compound of formula (I):

wherein R is selected from the group consisting of hydroxyl, halogen,alkyl, alkoxy, and amino and n is 0, 1, 2, 3, 4, 5, or 6.

Any of the aromatic compounds of formula (I) described herein can beused in a method of producing a non-human animal model of a cornealectatic disease. In preferred embodiments, the aromatic compound offormula (I) is benzene or phenol.

Any of the methods for applying an aromatic compound of formula (I) to acornea of an eye of a non-human animal described herein with referenceto producing a non-human animal model of corneal angiogenesis accordingto the invention can be used in a method of producing a non-human animalmodel of a corneal ectatic disease according to the invention. Preferredmethods of application include topical application, and preferredtopical compositions include liquid compositions, such as aqueoussolutions or other aqueous vehicle. In a preferred embodiment, anaqueous solution or vehicle is applied to the cornea by eye drops.

According to embodiments of the invention, an effective amount of acompound of formula (I) needed to produce a non-human animal model of acorneal ectatic disease is about 30 μmol to 85 μmol, such as 30 μmol, 40μmol, 50 μmol, 60 μmol, 65 μmol 70 μmol, 75 μmol, 80 μmol, or 85 μmol.In general, an effective amount of an aromatic compound of formula (I)for producing a non-human animal model of a corneal ectatic disease isgreater than an effective amount of an aromatic compound of formula (I)for producing a non-human animal model of corneal angiogenesis. Theeffective amount can be applied in a single application or in multipleapplications, and is preferably applied in a single application.

In a particular embodiment of the invention, about 70 μmol to 85 μmol ofbenzene (i.e., about 5.5 mg to about 7.0 mg benzene) can be applied tothe eye of a non-human animal to produce a model of corneal keratoconus.For example, applying a 7.0 M to 8.5 M benzene in an aqueous vehiclesuch as in the form of eye drops can induce a structural distortion ofthe cornea, resulting in a model of corneal keratoconus. See FIG. 3. Inanother particular embodiment, about 30 μmol to 85 μmol of phenol can beapplied to the eye of a non-human animal to produce a model of cornealkeratoconus.

In a preferred embodiment of the invention, the corneal ectatic diseaseis corneal keratoconus. Corneal keratoconus is one of the more commoncorneal ectatic diseases that develops in humans. According toembodiments of the invention, in a non-human animal model of cornealkeratoconus, the structural distortion of the cornea can becharacterized as a change in shape from a rounded shape of the cornea toa conical shape. See, e.g., FIG. 3. As corneal keratoconus developsfollowing application of an aromatic compound of formula (I), the corneabegins to thin and protrude outward from the eye, creating a bulge.

According to particular embodiments, an albino animal can be used in amethod of the invention for producing a non-human animal model of acorneal ectatic disease.

The invention also relates to non-human animal models of corneal ectaticdiseases produced by a method of the invention. According to embodimentsof the invention, a non-human animal model of a corneal ectatic diseasehas a cornea comprising a structural distortion characterized as achange in shape form a rounded shape to a conical shape.

To the best of the knowledge of the inventor, until now there was noknown animal model of corneal ectatic diseases, such as cornealkeratoconus. The invention thus satisfies this need by providing ananimal model of corneal ectatic diseases that is simple and inexpensiveto produce, and also does not require complicated equipment.

According to embodiments of the invention, a smaller amount of anaromatic compound of formula (I) is needed to induce cornealangiogenesis as compared to the amount needed to induce a cornealectatic disease in a method of producing a non-human animal model, asdescribed herein. For example, about 1 μmol to 70 μmol of benzene orphenol are sufficient for inducing corneal angiogenesis when applied tothe eye of a non-human animal, whereas about 70 μmol to 85 μmol ofbenzene, or 30 μmol to 70 μmol of phenol, are needed to induce a cornealectatic disease, such as corneal keratoconus. See, e.g., FIG. 2 and FIG.3.

Therefore, as an illustrative example, in a method of producing anon-human animal model of corneal angiogenesis, 10 μL of a 0.1 M to 7.0M benzene solution can be administered to an eye of a non-human animal,whereas in a method of producing a non-human animal model of a cornealectatic disease, 10 μL of a 7.0 M to 8.5 M benzene solution can beadministered to an eye of a non-human animal. The concentration ofbenzene can also be varied to adjust the rate at which cornealangiogenesis is induced, and to adjust the amount of new blood vesselgrowth. See FIG. 2, which demonstrates that the amount of blood vesselgrowth observed in rat corneas increases as the concentration of benzeneapplied increases. Of course, one of ordinary skill in the art willreadily appreciate that there can be biological variability betweenspecies of the same animal in both the angiogenic response anddevelopment of corneal keratoconus or other corneal ectatic diseasesobserved.

Because corneal angiogenesis can be induced by a smaller amount of anaromatic compound of formula (I) than the amount needed to inducecorneal ectatic diseases, animal models of corneal ectatic diseases canalso comprise one or more new blood vessels in the cornea of the eye aswell as the structural distortions of the cornea observed uponapplication of high concentrations of an aromatic compound of formula(I). This is because when higher concentrations of the aromaticcompounds of formula (I) are used, new blood vessels typically grow inaddition to causing a structural distortion of the cornea.

According to embodiments of the invention, an anesthetic agent can beadministered to the eye of a non-human animal in addition to an aromaticcompound of formula (I) in a method of producing a non-human animalmodel of corneal angiogenesis or a corneal ectatic disease. Ananesthetic agent is a drug that causes an analgesic effect, i.e.,induces the absence of pain and/or sensation. Any anesthetic agent usedin surgical eye procedures can be used, including topical, local, andgeneral anesthetic agents. Preferably, a local anesthetic agent is used.Examples of anesthetic agents that can be used with the inventioninclude, but are not limited to, xylocaine, paracaine, tetracaine,bupivacaine, and lidocaine. The anesthetic agent can be administered byany method known in the art for administering anesthetic agents forsurgical eye procedures, such as by topical application. The anestheticagent can also be administered to the eye in the same composition as thearomatic compound of formula (I), e.g., aqueous solution.

A purpose of administering an anesthetic agent is to reduce any pain ordiscomfort associated with application of the aromatic compound offormula (I). The anesthetic agent can be administered to the eye beforethe aromatic compound of formula (I), simultaneously with theapplication of the aromatic compound of formula (I), or after thearomatic compound of formula (I) is applied, and is preferably appliedbefore or simultaneously with application of the aromatic compound offormula (I).

In other general aspects, the invention relates to methods of studyingcorneal angiogenesis and corneal ectatic diseases (e.g., keratoconus)using non-human animal models of the invention. For example, thenon-human animal models of the invention can be used to screen compoundsthat modulate corneal angiogenesis to identify modulators of cornealangiogenesis that can be used to treat or prevent diseases associatedwith increased or decreased in vivo angiogenesis. The non-human animalmodels of the invention can also be used to screen compounds that areeffective in treating or preventing corneal ectatic diseases, such ascorneal keratoconus.

According to embodiments of the invention, a method of screeningcompounds to modulate corneal angiogenesis or a corneal ectatic diseasecomprises:

-   -   (i) preparing a non-human animal model of corneal angiogenesis        or of a corneal ectatic disease comprising applying to at least        one eye of a non-human animal an effective amount of an aromatic        compound of formula (I);    -   (ii) administering a test compound to the at least one eye of        the non-human animal model; and    -   (iii) determining an effect of the test compound on at least one        of blood vessel growth in a cornea and structural distortion of        a cornea.        Any of the methods described herein can be used to produce a        non-human animal model for use in a method of screening a        compound according to the invention.

According to embodiments of the invention, a test compound can beadministered prior to, at the same time as, or after application of thearomatic compound of formula (I). A test compound can be administered byany method known in the art, including, but not limited to topicalapplication and injection. Preferably, test compounds are administeredtopically, such as in a liquid composition, solution, or aqueousvehicle. In certain embodiments, a test compound and an effective amountof an aromatic compound of formula (I) are administered together in asingle composition. The test compound and the effective amount of anaromatic compound of formula (I) can be administered to one eye of thenon-human animal, and the effective amount of the aromatic compound offormula (I) can be administered to the other eye. By comparing the eyetreated with the test compound to the eye not treated with the testcompound, the effects of the test compound on corneal angiogenesisand/or corneal ectatic diseases can be determined.

As used herein, the term “modulate” means having an effect on the onset,occurrence of, or progression of corneal angiogenesis or a cornealectatic disease. In one embodiment, modulate refers to inhibiting theonset of, slowing the progression of, or ameliorating one or more signsor symptoms of corneal angiogenesis or a corneal ectatic disease. In oneparticular embodiment, modulate refers to increasing or decreasingcorneal angiogenesis, e.g. the growth of new blood vessels. In anotherparticular embodiment, modulate refers to reducing or inhibiting thedevelopment of structural distortion, or “loss of shape” of the cornea.

According to embodiments of the invention, modulators of cornealangiogenesis can be useful for treating or preventing cornealangiogenesis by, for example, reducing or eliminating the growth of oneor more new blood vessels, and can also be useful in treating orpreventing other diseases associated with in vivo angiogenesis.Modulators of corneal ectatic diseases can be useful for treating orpreventing corneal ectatic diseases, such as corneal keratoconus.

According to embodiments of the invention, when a test compound isapplied to the eye prior to or subsequent to application of the aromaticcompound of formula (I), the time between applications can vary from afew minutes, to a few hours, to a few days, depending on the particulartest compound, its mode of action, its efficacy, etc. For example, atest compound can be applied to one eye, and then an aromatic compoundof formula (I) can be applied to both eyes 24 hours later. As anotherexample, an aromatic compound of formula (I) can be applied to botheyes, and then a test compound can be applied three weeks later afterthe onset of corneal angiogenesis is observed. One of ordinary skill inthe art will be able to appropriately design a screening experimentdepending on the desired objective.

According to embodiments of the invention, the effect of a test compoundon modulating corneal angiogenesis and/or corneal keratoconus can bedetermined by any method known in the art in view of the presentdisclosure. For example, the effect can be determined by visualobservation or histological studies. Histological studies can beperformed by treating a section of a cornea with a dye that stains bloodvessels, and observing under a microscope. An exemplary dye that can beused for histological studies is hematoxylin and eosin stain.

The invention also relates to a method of identifying substances harmfulto human health. According to embodiments of the invention, the methodcomprises:

-   -   (i) applying a test substance to a cornea of at least one eye of        a non-human animal; and    -   (ii) determining an effect of the test substance on at least one        of blood vessel growth in the cornea and structural distortion        of the cornea.        Compounds or materials that induce corneal angiogenesis or        corneal ectatic diseases, such as corneal keratoconus, can be        harmful to human health. A substance that is “harmful to human        health” is one that is toxic, increases the incidence of fatal        diseases (e.g., cancer), or produces some other adverse        biological reaction, such as irritation, rash, etc. Therefore,        by screening a substance for its effect on corneal angiogenesis        and corneal ectatic diseases according to a method of the        invention, a substance that is harmful to health or that poses        significant health risks to mammals, such as humans, can be        identified.

According to embodiments of the invention, a method of identifyingsubstances harmful to human health can be used to screen substances usedin any industry including, but not limited to, the food industry, thepharmaceutical industry, and the electronics industry. Any substance canbe screened, such as a compound, material, solvent, etc. According toembodiments of the invention, the test substance is applied to a corneaof an eye of a non-human animal using any method known in the art inview of the present disclosure, including injection or topicalapplication, such as by drops or by swabbing.

Without wishing to be bound by any theories, one possible explanationfor the observed effect of aromatic compounds of formula (I), such asbenzene and phenol, on blood vessel growth is summarized as follows. Theintrinsic ability of melanin, and its derivatives, analogs, andvariants, to split the water molecule into hydrogen and oxygen uponabsorption of electromagnetic energy, such as light energy, haspreviously been reported in U.S. Pat. No. 8,455,145. It is also knownthat high levels of oxygen have an anti-angiogenic effect. It isbelieved that benzene and phenol can impair the function of melanocytes,which are the cells that produce melanin. Melanocytes are located, amongother places, in the middle layer of the eye. By impairing the functionof melanocytes, the amount of melanin produced is significantly reduced,and thus the amount of oxygen produced by melanin is also reduced,decreasing oxygen levels and promoting angiogenesis. On the other hand,it is believed that when melanin is more abundant, the dissociation ofwater molecules occurs more rapidly and/or more readily, resulting in ahigher partial pressure of oxygen, consequently reducing the amount ofangiogenesis. Accordingly, it is believed that reduced levels of melaninstimulate angiogenesis, and increased levels of melanin inhibit, or atleast significantly decrease, angiogenesis.

Again without wishing to be bound by any theories, it is hypothesizedthat a smaller amount of a compound of formula (I) is needed to inducecorneal angiogenesis than corneal ectatic diseases, because a greaterinhibition of melanin production is needed to induce the onset ofcorneal ectatic diseases. In contrast, less inhibition of melaninproduction is believed to be sufficient to cause corneal angiogenesis.This hypothesis is based on the observation that corneal angiogenesiscan be induced with lower concentrations of aromatic compounds offormula (I), as compared to the concentrations of aromatic compounds offormula (I) needed to induce corneal keratoconus as discussed in moredetail in the examples below.

This invention will be better understood by reference to thenon-limiting examples that follow, but those skilled in the art willreadily appreciate that the examples are only illustrative of theinvention and are not to be construed as limiting the invention in anyway.

EXAMPLES Example 1 Rat Model of Corneal Angiogenesis and Corneal EctaticDisease Produced with Phenol

Rat models of corneal angiogenesis and corneal keratoconus were producedas follows. Aqueous solutions of phenol having a concentration of 0.1 M,0.5 M, 1.0 M, 3.0 M, 5.6 M, or 8.5 M were prepared by mixing phenol andwater. The solutions were then sterilized by heating to 100° C. for 15minutes. Then, 10 μL of the sterile phenol solution was topicallyapplied to the center of the cornea of the right eye of a Wistar ratthat was two months old. Five rats were treated for each concentrationof phenol tested. The phenol solution was allowed to absorb into theeye, and was applied only once. Because the eye has its own naturalprotective mechanisms, it was not necessary to wash the eye at the endof the application.

Prior to application of the phenol solution, there were no visible bloodvessels in the cornea (FIG. 1A). Instead, the vessels seen are in theiris of the eye. However, new blood vessels began to form in the corneaupon treatment with all concentrations of the phenol solution tested inat least one of the rats in each group one week after application of theaqueous phenol solution. The amount of new blood vessels observed in thecornea was even greater three weeks after application (FIG. 1B,application of 3 M solution). Blood vessel growth was observed for aperiod of fourth months. As shown in FIG. 1B, profuse vascularizationbegan in the sclero-corneal limbus and progressed towards the center ofthe corneal surface. Except for the new corneal vessels, no otheranatomical or inflammatory alterations were observed. The vessels of theiris were deeper than those that grew in the cornea, and are delimitedby the pupil border. At the higher concentrations of phenol tested (3.0M, 5.6 M, and 8.5 M), changes associated with corneal ectasia,particularly corneal keratoconus, were also observed in some of therats. The results are summarized in Table 1 below.

TABLE 1 Preparing rat models of corneal angiogenesis and cornealkeratoconus with phenol in rats. Observation in Treated Eye (Number ofRats) Phenol Number of Corneal No Concentration Rats TreatedAngiogenesis Keratoconus Change 0.1M 5 1 0 4 0.5M 5 1 0 4 1.0M 5 3 0 23.0M 5 4 2 1 6.5M 5 4 2 1 8.5M 5 3 1 2

Histological studies were performed after eight weeks following theapplication of phenol. The cornea was excised, and a piece of thecorneal epithelium was stained with hematoxylin and eosin stain. Asshown in FIG. 1C, well differentiated blood vessels can be seen underthe basal membrane of the corneal epithelium (pointed to by arrow 2).

The results of the experiment shown above in Table 1 demonstrate thatphenol applied directly to the eye of a rat induces cornealangiogenesis, and that animal models of corneal angiogenesis can beproduced by applying phenol to an eye of the animal. The results alsoindicate that when higher concentrations of phenol are applied, cornealectatic diseases are also induced.

Example 2 Rat Model of Corneal Angiogenesis Produced with Benzene

Rat models of corneal angiogenesis were produced as follows. Aqueousbenzene compositions having the desired concentration (between 3.0 M and7.0 M) were prepared by mixing benzene and water. The aqueouscomposition was then sterilized by heating to 100° C. for 15 minutes. Inparticular, 3.0 M, 4.0 M, 5.0 M, 6.0 M, or 7.0 M aqueous benzenecompositions were prepared. Then, 10 μL of each composition wastopically applied to the center of the cornea of the right eye of aWistar rat that was two to three months old. The benzene aqueouscomposition was allowed to absorb into the eye, and was applied onlyonce. Because the eye has its own natural protective mechanisms, it wasnot necessary to wash the eye at the end of the application.

Prior to benzene application, there were no visible blood vessels in thecornea, and the corneas of the rats appeared similar to that as shown inFIG. 1A. However, as shown in FIG. 2, new blood vessels began to form inthe cornea upon treatment with all concentrations of benzene as early as15 days after application. The amount of new blood vessels observed inthe cornea was even greater three weeks after application, and theobserved growth of new blood vessels persisted for at least four monthsfrom the initial application of benzene, at which time the eyes weresacrificed for histological studies.

The results of the experiment demonstrate that benzene applied directlyto the eye of a rat induces corneal angiogenesis, and that animal modelsof corneal angiogenesis can be produced by applying benzene to an eye ofthe animal. The results also indicate that when higher concentrations ofbenzene were initially applied, the amount of new blood vessels observedwithin 15 days of application also increased. Thus, the amount of newblood vessel growth in rats can depend upon the concentration of thearomatic compound, with higher concentrations able to more rapidlyinduce corneal angiogenesis as compared to lower concentrations. Theresults further indicate that the amount of corneal angiogenesisobserved increases over time following the initial application of thearomatic compound.

Example 3 Rat Model of Corneal Keratoconus

A rat model of corneal keratoconus was produced as follows. An aqueousbenzene composition having a concentration of 7.0 M to 8.5 M wasprepared by mixing water and benzene. The composition was thensterilized by heating the solution to 100° C. for 15 minutes. Theaqueous benzene composition (10 μL) was topically applied directly tothe center of the cornea of one eye of a Wistar rat by micropipetting.The rat was three months old, and benzene was applied only once.

In addition to angiogenesis (i.e., new blood vessel growth), the cornealtissue was characterized by marked thinning, and outward protrusion(FIG. 3). The observed changes in the cornea, which began to occurwithin the first week following benzene application, started as changesin corneal transparency, followed by the growth of new blood vessels inthe periphery of the cornea (i.e., angiogenesis), and finally protrusionof the cornea within the second week following application. The changeswere compatible with non-inflammatory corneal ectasia, and particularlycorneal keratoconus.

It should also be noted that when 10 μL of a 9.0 M aqueous benzenecomposition were applied to the eyes of Wistar rats according to thesame procedure, corneal keratoconus was so severe that the ratsdeveloped endophthalmitis (intraocular infection).

The results of the experiment indicate that applying benzene to the eyeof rats induces corneal keratoconus, and that animal models of cornealkeratoconus can be produced by administering aromatic compounds offormula (I) to an eye of the animal.

Example 4 Screening Compounds that Modulate Corneal Angiogenesis and/orCorneal Ectatic Diseases

Potential therapeutic agents (i.e., test compounds) are tested formodulation of corneal angiogenesis and corneal ectatic diseases. Anaqueous solution of an aromatic compound of formula (I) is prepared, andtopically applied in the form of eye drops to the cornea of both eyes ofa rodent. Then, a solution of a candidate therapeutic agent is topicallyapplied to the cornea of one eye of the rodent. The eyes of the rodentare observed, and the eye treated with the candidate therapeutic agentis compared to the untreated eye to determine the efficacy of the testcompound, if any, in modulating corneal angiogenesis or a cornealectatic disease. Visual observation and histological studies areperformed to determine the effects of the test compound.

Example 5 Identifying Substances Harmful to Human Health

Potentially harmful substances and materials (i.e., test substances) aretested for their effect on inducing corneal angiogenesis and cornealectatic diseases. The test substance is topically applied to a cornea ofone eye of the rodent. The eyes of the rodent are observed, and the eyetreated with the test substance is compared to the untreated eye todetermine the effect of the test substance, if any, in causing cornealangiogenesis or a corneal ectatic disease. Visual observation andhistological studies are performed to determine the effects of the testsubstance. Test substances that induce corneal angiogenesis or cornealectatic diseases are identified as potentially harmful to human health.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of theinvention as defined by the appended claim.

I claim:
 1. A method of producing a non-human animal model of cornealangiogenesis, the method comprising applying to a cornea of at least oneeye of a non-human animal an effective amount of an aromatic compoundselected from the group consisting of phenol and benzene, wherein theeffective amount of the aromatic compound applied is about 1 μmol toabout 70 μmol, and the effective amount of the aromatic compound isapplied as a composition having a concentration of 0.1 M to 8.5 M. 2.The method of claim 1, wherein the aromatic compound is phenol.
 3. Themethod of claim 1, wherein the non-human animal is a rodent.
 4. Themethod of claim 1, further comprising administering an anesthetic agentto the at least one eye.
 5. A non-human animal model of cornealangiogenesis prepared by the method of claim 1, wherein the non-humananimal model has substantially no fibrosis in the cornea.
 6. A method ofscreening compounds to modulate corneal angiogenesis, the methodcomprising: (i) preparing a non-human animal model of cornealangiogenesis by a method comprising applying to a cornea of at least oneeye of a non-human animal an effective amount of an aromatic compoundselected from the group consisting of benzene and phenol, wherein theeffective amount of the aromatic compound applied is about 1 μmol toabout 70 μmol, and the effective amount of the aromatic compound isapplied as a composition having a concentration of 0.1 M to 8.5 M; (ii)administering a test compound to the at least one eye of the non-humananimal model; and (iii) determining an effect of the test compound onblood vessel growth in the cornea.
 7. The method of claim 6, wherein thetest compound is administered to the at least one eye prior to,simultaneously with, or after application of the aromatic compound. 8.The method of claim 6, wherein the aromatic compound is phenol.
 9. Themethod of claim 2, wherein the effective amount of phenol applied isabout 1 μmol to about 10 μmol.
 10. The method of claim 1, wherein thearomatic compound is benzene.
 11. The method of claim 10, wherein theeffective amount of benzene applied is about 30 μmol to about 70 μmol.12. The method of claim 8, wherein the effective amount of phenolapplied is about 1 μmol to about 10 μmol.
 13. The method of claim 6,wherein the aromatic compound is benzene.
 14. The method of claim 13,wherein the effective amount of benzene applied is about 30 μmol toabout 70 μmol.
 15. The method of claim 1, wherein the effective amountof the aromatic compound is applied as an aqueous vehicle.
 16. Themethod of claim 2, wherein the effective amount of phenol is applied asan aqueous vehicle.
 17. The method of claim 10, wherein the effectiveamount of benzene is applied as an aqueous vehicle.
 18. The method ofclaim 6, wherein the effective amount of the aromatic compound isapplied as an aqueous vehicle.
 19. The method of claim 8, wherein theeffective amount of phenol is applied as an aqueous solution.
 20. Themethod of claim 13, wherein the effective amount of benzene is appliedas an aqueous solution.